Figure 5.
Evaluation of proximal signaling in VLK-deficient platelets. (A) Representative image of p-Akt (S473) and total Akt levels in lysates from CTL and cKO platelets resting or stimulated with 100 µM AYPGKF. (B) Cumulative percent phosphorylation of S473 in Akt; n = 3 independent experiments. (C) Representative image of p-Erk1/2 (T202/Y204) and Erk1/2 total levels in lysates from CTL and cKO platelets resting or stimulated with 100 µM AYPGKF. (D) Cumulative percent phosphorylation of Erk1/2; n = 3 independent experiments. (E) Representative image of p-Akt (S473) and total Akt levels in lysates from CTL and cKO platelets resting or stimulated with 100 µM AYPGKF and/or 2.5 µM ADP. (F) Cumulative percent phosphorylation of S473 in Akt; n = 3 independent experiments. (G) Representative image of p-Erk1/2 (T202/Y204) and Erk1/2 total levels in lysates from CTL and cKO platelets resting or stimulated with 100 µM AYPGKF and/or 2.5 µM ADP. (H) Cumulative percent phosphorylation of Erk1/2; n = 3 independent experiments. (I) Proposed model for the role of VLK in platelet function. Resting platelet: Proteins that are tyrosine phosphorylated by VLK in the megakaryocyte secretory pathway during thrombopoiesis are present in extracellular domains of surface proteins or intragranular proteins of resting platelets. VLK is localized to α-granules. ADP and ATP are concentrated in dense granules. Activated platelet: Activation with a submaximal agonist results in partial activation of kinases involved in dense granule release, such as Akt and Erk1/2. Signaling downstream of Akt and Erk1/2 results in release of ADP from dense granules. VLK appears to act in these steps leading to dense granule release by mechanisms that are yet to be determined but could include phosphorylation of receptors on extracellular domains, phosphorylation of secretory chaperones, or phosphorylation of dense granule resident proteins important for release. ADP released from dense granules stimulates purinergic receptors, enhancing signaling through kinases involved in granule release. Agonist stimulation also causes exocytosis of α-granules, resulting in the release of VLK into the extracellular environment along with ATP and VLK substrates.

Evaluation of proximal signaling in VLK-deficient platelets. (A) Representative image of p-Akt (S473) and total Akt levels in lysates from CTL and cKO platelets resting or stimulated with 100 µM AYPGKF. (B) Cumulative percent phosphorylation of S473 in Akt; n = 3 independent experiments. (C) Representative image of p-Erk1/2 (T202/Y204) and Erk1/2 total levels in lysates from CTL and cKO platelets resting or stimulated with 100 µM AYPGKF. (D) Cumulative percent phosphorylation of Erk1/2; n = 3 independent experiments. (E) Representative image of p-Akt (S473) and total Akt levels in lysates from CTL and cKO platelets resting or stimulated with 100 µM AYPGKF and/or 2.5 µM ADP. (F) Cumulative percent phosphorylation of S473 in Akt; n = 3 independent experiments. (G) Representative image of p-Erk1/2 (T202/Y204) and Erk1/2 total levels in lysates from CTL and cKO platelets resting or stimulated with 100 µM AYPGKF and/or 2.5 µM ADP. (H) Cumulative percent phosphorylation of Erk1/2; n = 3 independent experiments. (I) Proposed model for the role of VLK in platelet function. Resting platelet: Proteins that are tyrosine phosphorylated by VLK in the megakaryocyte secretory pathway during thrombopoiesis are present in extracellular domains of surface proteins or intragranular proteins of resting platelets. VLK is localized to α-granules. ADP and ATP are concentrated in dense granules. Activated platelet: Activation with a submaximal agonist results in partial activation of kinases involved in dense granule release, such as Akt and Erk1/2. Signaling downstream of Akt and Erk1/2 results in release of ADP from dense granules. VLK appears to act in these steps leading to dense granule release by mechanisms that are yet to be determined but could include phosphorylation of receptors on extracellular domains, phosphorylation of secretory chaperones, or phosphorylation of dense granule resident proteins important for release. ADP released from dense granules stimulates purinergic receptors, enhancing signaling through kinases involved in granule release. Agonist stimulation also causes exocytosis of α-granules, resulting in the release of VLK into the extracellular environment along with ATP and VLK substrates.

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